Tuberculosis in alpaca (Lama pacos) on a farm in Ireland. 1. A clinical report

This case report describes tuberculosis (TB) due to infection with Mycobacterium bovis (M. bovis) in alpaca (Lama pacos) on a farm in Ireland. Two severely debilitated alpaca were presented to the University Veterinary Hospital, University College Dublin in November 2004. Bloods were taken, and haematology and biochemistry results were indicative of chronic infection. Radiological examination showed evidence of diffuse granulomatous pneumonia suggestive of tuberculosis. On necropsy there were granulomatous lesions present throughout many body organs including lung, liver, kidney, intestine as well on peritoneum and mesentery. Culture of acid-fast bacilli from lesions led to a diagnosis of tuberculosis due to M. bovis. The use of intradermal skin testing proved inefficient and unreliable for ante mortem diagnosis of tuberculosis in alpaca. Infection due to M. bovis should be considered among the differential diagnoses of debilitating diseases in alpaca, particularly those farmed in areas known to be traditional black spots for tuberculosis in cattle.     

Serological survey of Encephalitozoon cuniculi in farm rabbits in Italy

Rabbit sera (n = 1600) from 40 commercial farms were submitted to a serological screening for Encephalitozoon cuniculi by an enzyme-linked immunosorbent assay (ELISA) and a carbon immunoassay (CIA test). Antibodies anti-Encephalitozoon cuniculi were found in 505/1600 (31.6%) sera analysed, and all the farms (100%) resulted positive. Rabbits older than 4 months showed a significantly higher seropositivity for E. cuniculi (chi-squared test: p < 0.0001) than rabbits under 4 months, E. cuniculi sero-prevalence showed an increasing trend in rabbits within the farm along with the increase in the “number of rabbits on the farm”; however, this trend was not significant (Spearman’s correlation: p = 0.073).The findings of the present study confirm that rabbit is the main reservoir of E. cuniculi; they are of epidemiological relevance and immediate public health importance because of the recognized infectivity in humans by the microsporidium.     

Efficacy of strain RB51 vaccine in protecting infection and vertical transmission against Brucella abortus in Sprague-Dawley rats

Immunizing animals in the wild against Brucella (B.) abortus is essential to control bovine brucellosis because cattle can get the disease through close contact with infected wildlife. The aim of this experiment was to evaluate the effectiveness of the B. abortus strain RB51 vaccine in protecting infection as well as vertical transmission in Sprague-Dawley (SD) rats against B. abortus biotype 1. Virgin female SD rats (n = 48) two months of age were divided into two groups: one group (n = 24) received RB51 vaccine intraperitoneally with 3 × 10¹⁰ colony forming units (CFU) and the other group (n = 24) was used as non-vaccinated control. Non-vaccinated and RB51-vaccinated rats were challenged with 1.5 × 10⁹ CFU of virulent B. abortus biotype 1 six weeks after vaccination. Three weeks after challenge, all rats were bred. Verification of RB51-vaccine induced protection in SD rats was determined by bacteriological, serological and molecular screening of maternal and fetal tissues at necropsy. The RB51 vaccine elicited 81.25% protection in SD rats against infection with B. abortus biotype 1. Offspring from rats vaccinated with RB51 had a decreased (p < 0.05) prevalence of vertical transmission of B. abortus biotype 1 compared to the offspring from non-vaccinated rats (20.23% and 87.50%, respectively). This is the first report of RB51 vaccination efficacy against the vertical transmission of B. abortus in the SD rat model.     

鉴别犬瘟热病毒疫苗株和野毒株RT—PCR—RFLP方法的建立及应用

根据Onderstepoort株H基因序列,设计1对引物建立RT-PCR-RFLP检测方法,对不同宿主来源的疑似犬瘟热临床样品进行检测,并对检出的犬瘟热病毒野毒山东株PCR产物进行克隆和序列分析,验证RT—PCR—RFLP检测方法。结果RT—PCR扩增片段为1921bp,产物经RFLP分析,野毒株的PCR产物能被NdeI酶切为1282、345和294bp3个片段,弱毒疫苗株则不能被切开;病毒RNA的最小检出量为2．15ng。临床检测共检出19份样品为犬瘟热阳性,其中15份为野毒株感染,其H基因编码区全长为1824bp,均在1279和1543处有NdeI酶切位点,推导氨基酸与野毒株的同源性在92．9％～96．9％,与疫苗株的同源性在89．0％～90．8％,进化树分析显示这些毒株在基因型上属于Asia-1型,为野毒株谱系。该方法的建立为临床上犬瘟热病毒的鉴别检测和诊断提供了依据。     

头孢氨苄免疫层析表面增强拉曼光谱检测方法研究

【目的】 建立一种简便、快速、灵敏度高、特异性强的头孢氨苄免疫层析表面增强拉曼光谱(SERS)检测新方法。【方法】 制备金银复合核壳结构的纳米贵金属,用来标记头孢氨苄抗体和拉曼信号分子5,5’-二硫代双2-硝基苯甲酸 (DTNB),合成拉曼免疫探针,用透射电子显微镜和双光束紫外分光光度计对纳米金和修饰拉曼信号分子的金银复合核壳结构进行表征。将合成的拉曼免疫探针固定在微孔板上,待测样品中的头孢氨苄与包被在硝酸纤维素膜上的头孢氨苄抗原竞争结合拉曼免疫探针,通过免疫层析试纸条结合共聚焦拉曼光谱仪读取DTNB的信号值,建立头孢氨苄定量检测技术,并应用于实际牛奶样品的检测。【结果】 透射电子显微镜和双光束紫外分光光度计表征结果显示,纳米银成功包裹在金颗粒表面。用共聚焦拉曼光谱仪检测免疫层析试纸条上拉曼信号分子的特征峰,拉曼信号分子特征峰为1 062、1 154、1 334和1 558 cm^-1,其中1 334 cm^-1处特征峰信号最强,选取此处峰高定量测定头孢氨苄的含量。头孢氨苄免疫层析表面增强拉曼光谱检测的线性范围为0~1 ng/mL,检测限为0.001 ng/mL,头孢氨苄的半数抑制质量浓度为0.05 ng/mL。与头孢克洛交叉反应率为111.1%,与头孢曲松钠、头孢夫辛酯、头孢噻吩钠交叉反应率均低于0.1%。实际样品加标浓度为1、4和8 ng /mL,回收率分别为94.4%、103.3%和97.5%。【结论】 本研究建立的头孢氨苄免疫层析表面增强拉曼光谱检测方法操作简便、快速、灵敏,耗材成本低廉,可满足奶牛养殖农场和乳品加工企业进行大批量样品初筛检测需求。     

抗丝状支原体丝状亚种单克隆抗体的制备及初步应用

旨在制备抗丝状支原体丝状亚种(Mmm)的特异性单克隆抗体(MAb),为牛传染性胸膜肺炎(CBPP)病原诊断的免疫学方法提供特异性抗体。本研究利用生物信息学技术分析了Mmm国内分离株Ben-1不同传代株的全基因组序列,选取M0071蛋白作为研究对象。将原核表达的可溶性重组蛋白M0071(rM0071)作为免疫原免疫BALB/c小鼠,通过有限稀释法和间接ELISA方法筛选得到能稳定分泌抗rM0071蛋白的单克隆抗体的杂交瘤细胞株。进一步制备单抗腹水并纯化,利用Western blot方法对该单抗进行特异性鉴定,同时测定其抗体效价和抗体亚类。随后利用间接免疫荧光试验(IFA)评价该单抗对细胞感染Mmm的检测能力。结果表明成功获得1株单克隆细胞株3C4A1,将其分泌抗体命名为MAb 3C4A1。特异性结果表明,MAb 3C4A1能与Mmm的分离株和标准株发生特异性反应,而不与山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、牛鼻支原体、无乳支原体、牛支原体、leachii支原体和牛A型巴氏杆菌等发生反应。抗体亚类鉴定MAb 3C4A1属于IgG1亚类、轻链为κ链。经间接ELISA测定其抗体效价为1∶256 000。IFA试验结果表明,MAb 3C4A1仅与感染EBL细胞的Mmm发生绿色荧光反应,而与牛鼻支原体、无乳支原体、牛支原体感染的细胞不发生荧光反应,特异性良好。本研究制备的MAb 3C4A1具有良好的特异性和免疫反应性,可作为CBPP病原免疫学诊断的工具,为进一步研制CBPP病原鉴别诊断试剂盒提供了基础材料。     

利福平乙基纤维素微球在小鼠体内的组织分布

以有机相分散、溶媒扩散二步法制备了利福平乙基纤维素微球，粒径范围2.5-12.5μm，其中粒径2.5-5.0μm的约占63％。采用微生物测定法研究了利福平乙基纤维素微球静脉给药后，利福平在小鼠体内的组织分布，且与游离利福平在小鼠体内组织分布进行了比较，小鼠单剂量静脉注射游离利福平或利福平乙基纤维素微球（相当于利福平原药）10mg/kg，测定0.5、1、3、7、12、24、48、72、120、168h共10个时间点各组织中的药物浓度。结果表明，游离利福平给药组和利福平乙基纤维素微球给药组在12h内均以肝脏中药物浓度为最高，为9.96-22.41μg/g，在3h内微球给药组高于游离药物给药组，7、12h微球给药组的药物浓度迅速下降，低于游离药物给药组；肾脏的药物浓度在12h内微球给药组明显低于游离药物给药组，24－168h微球给药组的药物浓度下降迟缓，显著高于游离药物给药组直到168药物浓度始终是微球给药组高于游离药物给药组，12h游离药物给药组已不能检出药物含量，而微球给药组直到168h仍维持在5.44μg/g，接近游离药物给药组的最高峰值；血清中利福平浓度在48h已不能检出，而微球给药组在168h为1.33mg/L；心脏、用脏微球给药组和游离药物给药组的药物浓度变化无统计学意义，与游离药物相比，利福平微球明显提高了肺脏中的药物分布，降低了利福平对肝脏、肾脏的毒性，并显著延缓了药物代谢，说明利福平微球具有长效、靶向作用。     

猴痘病毒PCR检测方法的建立

从GenBank上调取猴痘病毒的基因序列,经过分析,找出了猴痘病毒的特异性靶基因序列F3L片段,人工合成猴痘病毒MPV(AF380138)F3L基因片段(48048-48509bp)并插入质粒,作为病毒检测的模拟阳性模板。根据该序列设计并合成PCR引物,对模拟的阳性模板进行扩增,结果能扩增出与目的片段大小一致的条带。建立了PCR检测方法,经条件优化后,对鸡痘、禽痘、羊痘等14种相似病毒核酸进行PCR扩增,发现具有良好的特异性。PCR方法能扩增出0.3pg的阳性模板,显示建立的PCR方法具有高效、快速、特异、灵敏的特点,可用于口岸猴痘病毒的检疫。     

新生牛血清促细胞生长活性检测方法的建立与应用

牛血清是医药生物技术产品中重要的原辅材料之一,做好牛血清的质量控制是提高生物制品质量的重要环节。本研究通过应用PK15、BHK21和ST三种兽用生物制品研制中常用的细胞作为载体,选用国内同等价位5个不同厂家生产的新生牛血清作为比较对象,采用细胞倍增时间测定法、细胞克隆形成率测定法、细胞三次连续传代培养法、MTT比色法和微量终点稀释法五种血清质量鉴定的方法进行比较,检测新生牛血清的促细胞生长活性,最终确定了MTT比色法作为便捷高效快速筛选新生牛血清的检测方法。